The present disclosure relates to novel compounds which inhibit indoleamine-2,3-dioxygenase (IDO), specifically indoleamine 2,3-dioxygenase 1 (IDOL), and tryptophan-2,3-dioxygenase (TDO). The disclosure also contemplates the use of such compounds to treat disease indications mediated by activity of IDO1 or TDO.
The essential amino acid tryptophan is degraded through the kynurenine pathway, of which the first and rate limiting step is catalyzed by heme-containing oxidoreductase enzymes, including indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), that convert tryptophan to N-formylkynurenine. Although these enzymes perform the same biochemical function, they share limited homology and their expression is compartmentalized in different locations of the body. Whereas IDO1 is expressed in placenta, gut, lungs, epididymis, lymph nodes and tumor cells, TDO expression is found mainly in the liver and the brain. IDO1 and TDO control tryptophan concentration, and also the balance of kynurenine pathway metabolites. Dysregulation of the kynurenine pathway or an imbalance in favor of kynurenine metabolites due to IDO1 and TDO activity leads to numerous disease indications related to immunosuppression.
The local depletion of tryptophan and the accumulation of kynurenine pathway metabolites due to dioxygenase activity induce immune tolerance and suppression. It has been shown in experiments concerning gut immunity, mammalian pregnancy, tumor immune evasion, chronic infection, neurological disorders, inflammatory and autoimmune diseases, etc., that expression of IDO can induce immune tolerance through suppression of T cells by depletion of tryptophan, an obligate amino acid for effector T cells. General control non-derepressible-2 kinase (GCN2) prevents T cell proliferation after detecting tryptophan depletion. Furthermore, kynurenine metabolites promote helper T cell conversion into regulatory T cells (Tregs), which are also responsible for immune suppression.
The human body houses ten times more bacterial cells than human cells and many of these bacterial cells comprise the human gut microbiota. Although these bacterial cells are distinguishable from the self, the human body must maintain immunological tolerance with respect to these bacteria. IDO-deficient mice had elevated baseline levels of immunoglobulin A (IgA) and IgG in the serum and increased IgA in intestinal secretions. These mutant mice expressing higher levels of natural secretory IgA were more resistant to intestinal colonization by Citrobacter rodentium and experienced significantly attenuated colitis due to C. rodentium. Distinct from disease resistance, IDO has also been shown to induce disease tolerance, the reduction of the impact of infection on host fitness. IDO1 knockout (KO) mice failed to exhibit LPS endotoxin tolerance, whereas LPS tolerant IDO1 expressing mice were able to mount a fully protective tolerance state when infected by LPS-expressing Salmonella enterica Typhimurium. These findings suggest that pharmacological modulation of IDO activity may provide solutions to dysregulation of intestinal immunity and to diseases caused to enteric pathogens.
Immunosuppression by IDO is also exemplified by maternal tolerance towards allogeneic fetuses. The general laws of tissue transplantation suggest that allogeneic mammalian conceptus should not survive. However, implications of IDO expression at the maternal-fetal interface suggest that IDO prevents immunologic rejection of allogeneic fetuses from the uterus. Dosing pregnant mice with 1-methyl-tryptophan (1-MT) resulted in rejection of allogeneic fetuses through a T cell-mediated response. Tryptophan catabolism by IDO1 appears to suppress immunological rejection by maternal T cells, allowing survival of allogeneic concepti. Maternal tolerance towards the fetus due to IDO1 expression suggests that IDO1/TDO inhibitory compounds may be of use in abortion or contraception.
HIV infection chronically induces IDO1 expression, resulting in chronic depletion of tryptophan and T cell dysfunction. Tryptophan depletion favors the development of Tregs over other CD4+ helper T cell subsets that offer protective immune functions. Constitutive expression of IDO1 continuously shifts the equilibrium of tryptophan metabolism towards kynurenines, inducing immunosuppression and allowing for progression of HIV infection. However, it has been demonstrated that IDO inhibition enhances the level of virus-specific CD8+ T cells and concomitantly reduces the number of virally infected macrophages in a mouse model of HIV. These lines of evidence suggest that IDO1 inhibitors, possibly in combination with other anti-retroviral agents, may provide utility in treatment of HIV disease.
Tumors, while normally under immune surveillance, have been shown to have the ability to express IDO1 to create a local microenvironment favorable for tumor growth and metastasis. Depletion of tryptophan and accumulation of kynurenines blocks proliferation of effector T cells and promotes the development of Tregs, inducing an immunosuppressed state in which tumors can evade normal immune mechanisms.
Although IFN-g exhibits anti-tumor properties, the cytokine has also been shown to be a potent inducer of IDO expression, and therefore may have limited effects in the immunosuppressive tumor microenvironment. Recent studies, however, have indicated that treatment of dendritic cells using selective IDO1 inhibitor epacadostat resulted in more potent activation of tumor associated antigen-specific T cells, along with an increase in production of both IFN-g and tumor cell lysis. Combinatorial therapy using an IDO inhibitor and anti-CTLA-4 or anti-PD-1/PD-L1 antibodies improved tumor control, IL-2 production and CD8+ T cell proliferation in a mouse model of melanoma compared to single agent therapy. Additionally, blocking IDO during chemo-radiation therapy increases the anti-tumor efficacy of such treatment by causing widespread deposition of C3 complement responsible for tumor destruction. These lines of evidence suggest that IDO1 inhibition can reverse tumor resistance and when used in combination with therapeutic agents may control tumor growth and metastasis.
Tryptophan degradation using tryptophan-2,3-dioxygenase (TDO) also influences tumor immune resistance in a manner similar to that catalyzed by IDO. TDO expressed by neurons and liver cells catabolizes tryptophan into kynurenine, which in turn functions as an endogenous ligand of human aryl hydrocarbon receptor (AHR) in an autocrine and paracrine fashion. Activation of AHR by TDO-derived kynurenine suppresses antitumor immune responses and promotes tumor cell survival and motility. Accordingly, it has been shown that TDO inhibition promotes tumoral immune rejection. Data from a series of 104 tumor cell lines shows that 20 tumors expressed only TDO2, 17 expressed only IDO1 and 16 expressed both. This suggests that a method of therapy involving dual inhibition of both IDO and TDO could be effective against a greater proportion of tumors.
Infectious diseases often trigger inflammation, which in turn can induce IDO activity. Infection by Epstein-Barr virus has been demonstrated to be able to induce IDO expression due to upregulation of TNF-α and IL-6 through p38/MAPK and NF-κB pathways in monocyte-derived macrophages. IDO suppression of T cell proliferation and impairment of CD8+ T cell cytotoxic function may be important in creating an immunosuppressive microenvironment for virus survival. In a mouse model, infection by influenza A virus stimulated IDO activity in the lungs and lung-draining mediastinal lymph nodes. In this mouse model, influenza-induced IDO activity in the lungs enhanced morbidity, slowed recovery, restrained effector T cell responses, and altered the repertoire of virus-specific memory CD8 T cells. Given the correlation between IDO activity and weakened host immunity, IDO inhibitors may be useful in combating infectious diseases.
Additionally, IDO has been implicated in non-infectious inflammatory diseases. IDO KO mice do not display spontaneous disorders of classical inflammation. Instead of eliciting generalized inflammatory reactions, small molecule inhibitors of IDO alleviate disease severity in the models of skin cancer promoted by chronic inflammation, and in models of inflammation-associated arthritis and allergic airway disease. IDO has also been implicated in autoimmune arthritis. IDO2 mediates production of autoreactive antibodies, but IDO2 KO mice have been shown to maintain their ability to mount productive antibody responses against model antigens. Very common autoimmune diseases include rheumatoid arthritis, type 1 diabetes, lupus, Hashimoto's thyroid disease, multiple sclerosis (MS), inflammatory bowel disease (IBD, which includes Crohn's disease and ulcerative colitis), celiac disease, and asthma. Therefore, IDO inhibitors may prove to be useful in the treatment of classical or autoimmune inflammatory diseases.
Studies have shown tryptophan catabolites to be of neurological significance. Tryptophan degraded through the kynurenine pathway produces metabolites that are neuroactive and neurotoxic. Kynurenine can be synthesized into kynurenic acid (KYNA) by kynurenine aminotransferases. KYNA has been shown to exert a non-competitive antagonistic effect on a7-nicotinic acetylcholine receptors and may offer protection against glutamate induced excitotoxicity. Also acting as a free radical scavenger, KYNA is generally understood to be a protective agent in neurodegenerative mechanisms. In a different branch of the kynurenine pathway, kynurenine can be converted to 3-hydroxykynurenine (3-HK) which undergoes auto-oxidation. 3-HK is generally considered to be neurotoxic due to the production of free radicals during auto-oxidation. 3-HK can also be converted to 3-hydroxyanthronilic acid (3-HA) which has similar oxidative reactivity as 3-HK, and can interfere with T cell survival. Downstream processing of 3-HA leads to production of quinolinic acid (QUIN). QUIN is a weak endogenous agonist of N-methyl-D-aspartate (NMDA) receptors and causes greatest excitotocity in regions of the brain rich in NMDA receptors. An imbalance in kynurenine metabolites reflected by higher concentrations of neurotoxic species may result in neurodegenerative disease indications. Because IDO and TDO are responsible for kynurenine production, inhibitors of these enzymes could be beneficial for neuropathic patients.
Alzheimer's disease (AD) is a chronic neurodegenerative disease that most commonly manifests in the elderly population and is characterized by progressive memory loss. Hallmarks of AD pathology include amyloid β (Aβ) plaques and phosphorylated tau-constituted neurofibrillary tangles, and the kynurenine pathway may play an important role in the neurodegenerative process. AD mice exhibit a greater density of TDO immune-density cells and an increased expression of TDO mRNA in the cerebellum. TDO co-localizes with QUIN, neurofibrillary tangles and amyloid deposits in the hippocampus of human AD brains. Furthermore, QUIN has been demonstrated to be capable of inducing tau phosphorylation in the human brain. Activated microglia in AD may produce excessive amounts of kynurenine pathway metabolites, including QUIN, in response to phosphorylated tau and Aβ plaques, resulting in a progressive disease cycle. These lines of evidence suggest that increased tryptophan catabolism through the kynurenine pathway may be responsible for Aβ pathology, and inhibitors of TDO or IDO could be useful in halting disease progression.
Parkinson's disease (PD) is a neurodegenerative disorder that impairs the motor system. PD is characterized by loss of dopaminergic neurons and neuroinflammation, which can occur several years before the onset of symptoms. Activated microglia can utilize the kynurenine pathway to generate neuroactive compounds. In PD, QUIN production by microglia is increased, leading to excitotoxicity by acting as a NMDA agonist. KYNA is a neuroprotective tryptophan catabolite, but its synthesis by astrocytes is concomitantly decreased in PD. PD is associated with an imbalance between these two branches of the kynurenine pathway within the brain, and pharmacological modulation of this pathway may be a new therapeutic strategy to treat the disease.
Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by expansion of CAG repeats in the HD gene on chromosome 4. HD is associated with loss of muscle coordination and cognitive decline. Evidence of increased ratio of kynurenine to tryptophan in the peripheral blood plasma of human patients with HD suggests a possible role of abnormal tryptophan metabolism in contributing to neuronal dysfunction and damage in HD. Gene expression analysis of YAC128 mouse model of HD reveals increased striatal-specific Ido1 mRNA. Further studies continue to examine the role of kynurenine pathway in HD, showing that the striatum of IDO KO mice is less sensitive to NMDA receptor-mediated excitotoxicity induce by QUIN compared to wild-type littermate controls. Although activity of TDO is generally thought to be limited to the liver, ablation of TDO2 is neuroprotective in a Drosophila model of HD. These findings implicate dysregulation of tryptophan catabolism in HD neuropathology and suggest that IDO or TDO could be therapeutic targets in cases of HD.
Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a neurodegenerative disease that specifically targets neurons that control voluntary muscle movement. Symptoms of ALS include varying degrees of muscle stiffness and weakening, but the long term prognosis can be bleak, and often fatal. Although ALS is a multifactorial disease and its exact mechanism of pathology is yet to be understood, tryptophan catabolites have been implicated in ALS studies. Compared to samples from control subjects, cerebral spinal fluid (CSF) and serum samples of ALS patients show elevated levels of L-kynurenine and QUIN, and decreased levels of neuroprotective species picolinic acid (PIC). Furthermore, the neurons and microglia of the ALS motor cortex and spinal cord express greater levels of IDO and QUIN, implicating neuroinflammation and kynurenine pathway involvement in ALS. A separate study reveals that CSF samples of patients with bulbar onset of ALS contained higher levels of KYNA compare to those of patients with severe clinical status, suggesting a neuroprotective role of KYNA against excitotoxicity in ALS. Involvement of kynurenines in ALS has been brought to attention, and inhibition of IDO or TDO responsible for synthesis of neurotoxic kynurenines may be a new option for therapeutic intervention.
Multiple sclerosis (MS) is a complex autoimmune disease driven by Th1 cells targeting oligodendrocytes and the myelin sheath, resulting in an inflammatory response that leads to the formation of sclerotic plaques in the central nervous system. Early research on kynurenine pathway involvement in MS shows that patients with chronic disease have lower levels of tryptophan in serum and CSF samples, suggesting activation of the kynurenine pathway. Ex vivo CSF samples of human MS patients indicate a possible correlation between KYNA and disease progression: induction of the kynurenine pathway in early active phases of MS leads to increased KYNA production but later shifts to a decrease in KYNA levels, causing the kynurenine pathway to exert neurotoxic effects. Activated macrophages and microglia have been shown be present along the boundaries of MS lesions, and may be able to produce QUIN at concentrations sufficient to induce brain cell death. In the autoimmune encephalomyelitis (EAE) mouse model of MS, inhibition of IDOL using 1-methyl-tryptophan has been shown to exacerbate disease status and allow proliferation of T-cell responses. Because the various branches of the kynurnine pathway can produce either neurotoxic or neuroprotective tryptophan catabolites, it is unclear whether activation of the pathway is beneficial in MS treatment. However, modulation of the kynurenine pathway may still be a valid strategy to treat MS.
Tryptophan degradation has also been implicated in neuropsychiatric disorders. An imbalanced kynurenine pathway may be a pathophysiological promoter in schizophrenia: CSF samples of schizophrenic patients contain higher ratios of KYNA to QUIN compared to controls, possibly due to compromised function of enzymes involved in QUIN synthesis. Since KYNA is an antagonist of the NMDA receptor, while QUIN is an agonist, a shift in this ratio may be reflected in the behavioral domain. A single nucleotide polymorphism in kynurenine 3-monooxygenase (KMO), one enzyme responsible for QUIN production, correlates with decreased KMO expression and increased CSF KYNA levels, and may be responsible for lifetime psychotic features in bipolar disorder patients.
Tryptophan can also be converted to 5-hydroxytryptamine (5-HT) and later into serotonin and then melatonin. Depletion of tryptophan can cause episodes of depression, and IDO activity in the kynurenine pathway can decrease serotonin and trigger depression. In inflammation-associated depression, tryptophan catabolites can trigger the mood swing independently of serotonin. Conversion of tryptophan into kynurenine and later QUIN and 3HK is neurotoxic, and can induce a depressive state. Although the mechanisms of neuropsychiatric disorders differ from those of inflammation-associated neurodegenerative disorders, new methods of therapy may still involve modulation of the kynurenine pathway.
There has also been evidence of the kynurenine pathway influencing cardiovascular health. Especially in patients with end-stage renal disease, induction of IDO activity and consequent increase in serum kynurenines lead to a number of cardiovascular complications. Kynurenines have been associated with hyperfibrinolysis, which has been causally related to the development of atherosclerosis. Elevated levels of kynurenine, QUIN, matrix metalloproteinases (MMPs) and a tissue inhibitor of MMPs have been discovered in continuous ambulatory peritoneal dialysis patients with cardiovascular disease (CVD) than patients without CVD and controls. Additionally, it has been demonstrated that QUIN is positively correlated with MMP-2 and the tissue inhibitor of MMP-2, which are responsible for the degradation of the extracellular matrix components involved in vascular wall remodeling. These lines of evidence suggest a connection between activation of the kynurenine pathway and cardiovascular disease prevalence in patients with chronic kidney disease. Given the above discussion of disease indications relating to dysregulation of tryptophan catabolism, there exists a strong unmet need for new compounds that inhibit IDO or TDO, two enzymes that are responsible for activation of the kynurenine pathway and tryptophan depletion. Development of TDO and IDO inhibitors and methods of treatment using such inhibitors is a key step in combating the aforementioned diseases and disorders.
There has been a considerable amount of effort towards making new IDO1 and TDO inhibitors for human use since the discovery of indoleamine 2,3-dioxygenase 1 as an important target for anticancer therapy in 2003. However, only a few potent IDO1 inhibiting compounds have entered clinical trials, and none have been approved by the FDA as of date.
Accordingly, there remains a strong unmet need for new IDO1 and TDO inhibiting compounds.